Microfluidic flow assay and methods of use

ABSTRACT

A method for evaluating a blood product of an individual are provided. Specifically, a method to utilize a microfluidic flow assay, which includes a substrate surface comprising lipid coated particles and microfluidic channels through which a blood product can flow. The lipid coated particles comprise functional molecules that can induce or inhibit the coagulation cascade.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. of patent application Ser. No.13/929,141, filed on Jun. 27, 2013, which issued as U.S. Pat. No.9,709,579 on Jul. 18, 2017, which claims the benefit of U.S. ProvisionalPatent Application Ser. No.61/665,177, filed Jun. 27, 2012. Thesereferences are hereby incorporated herein by reference in theirentirety.

TECHNICAL FIELD

The invention relates to a microfluidic-based flow assay and device foruse in analyzing bleeding and anticoagulation disorders, dosinganticoagulant drugs, tracking the effects of pharmacologicalinterventions on coagulation, and methods of making the same.

BACKGROUND OF INVENTION

Maintaining the balance between bleeding and thrombosis remains one ofthe greatest challenges facing the biomedical community. Excessivebleeding is an important medical issue. For example, post partumbleeding represents a leading cause of maternal mortality and causesserious morbidity in developing countries. Individuals with geneticbleeding disorders, such as hemophilia, have a decreased ability to clotblood because of deficiencies in certain coagulation factors.

On the other end of the spectrum, excessive clotting, or thrombosis, isa major complication of surgery and is integrally involved inatherosclerosis, obesity, infection, diabetes, cancer, and autoimmunedisorders. Over the last decade, significant advances have been made inunderstanding the molecular basis of bleeding and thrombotic disorders;however, a large portion of the observed variability remains unknown.

Parallel with these discoveries, there has been a rapid development ofnew drugs such as recombinant proteins for replacement andinterventional therapies. Interestingly, what remains strikinglydeficient in clinical hematology are techniques to diagnose a very broadrange of disorders of both deficient and excessive clotting as well asto monitor the effects of therapeutic interventions.

The formation of a stable fibrin network is necessary for hemostasis,which requires fibrinogen conversion to fibrin. In purified systemscontaining only thrombin and fibrinogen, it has been shown that fibrinpolymerization can only occur in a narrow set of conditions that aredefined by the rate of thrombin formation and the shear rate (Neeves etal., Biophysical Journal, 2010, 98; 1344-1352). Most coagulation assaysdo not account for the interplay between flow and surface reactions,which could affect clot properties like fiber thickness, fibrin clotheight, fiber alignment with flow, and resistance to lysis. Theseproperties can be useful in differentiating plasma clots of healthyindividuals from those with thrombotic or haemostatic disorders.

Diagnosing the severity of bleeding a disorder is impossible withcurrent bleeding assays, particularly because most current bleedingassays test for either platelet function and/or platelet coagulationusing whole blood, however, these assays do not allow for the useplasma. Additionally, most existing solutions do not properly create anenvironment which properly simulates a natural human wound or point ofbleeding. Also, most of these conventional assays occur under static, orno flow, conditions. Since blood is a moving fluid, however, there areseveral advantages to studying it under flow in bleeding diagnostics.

SUMMARY OF INVENTION

One embodiment of the invention relates to a microfluidic devicecomprising at least one microfluidic channel and at least one substratesurface in the microfluidic channel. The substrate surface comprises aplurality of lipid coated particle that are immobilized on the substratesurface. The lipid coated particles comprise at least one functionalmolecule that induces coagulation.

In one aspect, the substrate surface is functionalized glass.

In another aspect, the plurality of lipid coated particles comprises aplurality of particles having a hydrophilic surface. In one aspect, thelipid coated particles comprise one or more phospholipid structures. Thephospholipid structures can be selected from phosphotidylserine,phosphotidlcholine, phosphatidic acid, phosphatidylethanolamine,phophoinositides, phosphosphingolipids, and combinations thereof. Instill another aspect, the plurality of lipid coated particles isimmobilized to the substrate surface by an immobilization method. Theimmobilization can be selected from covalent bonding, electrostaticinteractions and hydrogen bonding. In yet another aspect, theimmobilized plurality of lipid coated particles is patterned to thesubstrate surface by a patterning method. The patterning method can beselected from microblotting and microstenciling. In another aspect, theimmobilized and patterned lipid coated particles are integrated into atleast one microfluidic channel.

In another aspect, the microfluidic device further compriseshydrodynamic focusing.

In still another aspect, the functional molecule of the coated lipidparticle of the device is one or more transmembrane proteins. Thetransmembrane proteins can be selected from tissue factor,thromobomodulin, endothelial cell protein C receptor, glycoproteinIIb/IIIa, glycoprotein VI, glycoprotein Ib/IX/V, P-selectin,glycoprotein IV, CD9, platelet endothelial cell adhesion molecule(PECAM-1), Ras-related protein 1b (rap1b), c-type lectin-like receptor 2(CLEC-2), intracellular adhesion molecule 1 (ICAM-1), intracellularadhesion molecule 2 (ICAM-2) and combinations thereof.

Another embodiment of the invention relates to a microfluidic devicemade by a method comprising providing a substrate, creating at least onesurface on the substrate, immobilizing and patterning a plurality oflipid coated particles onto the surface of the substrate. The lipidcoated particles are coated with lipid bilayers and comprise afunctional molecule that induces coagulation. The plurality of lipidcoated molecules is integrated into at least one microfluidic channel,which intersects at least a portion of the substrate surface.

Another embodiment of the invention relates to a plurality of lipidcoated particles made by a method comprising providing silica beads,making the silica beads hydrophilic, coating the surface of thehydrophilic silica beads with lipid bilayers and a functional molecule.The lipid bilayers comprise one or more phospholipid structures.

Yet another embodiment of the invention relates to a kit for measuringclotting characteristics of a blood product. The kit comprises ahermetically sealed microfluidic device, the microfluidic devicecomprising at least one microfluidic channel and at least one substratesurface provided in the at least one microfluidic channel, wherein theat least one substrate surface comprises a lipid coated particle whereinthe lipid coated particle comprises a functional molecule embedded inthe lipid, wherein the functional molecule induces coagulation.

A further embodiment of the invention relates to a method for evaluatinga blood product of an individual comprising perfusing the individual'sblood product over a microfluidic device under flow conditions tocontact the blood product with a functional molecule of a plurality ofcoated lipid particles, wherein the microfluidic device, comprises atleast one microfluidic channel; and at least one substrate surfaceprovided in the at least one microfluidic channel, wherein the at leastone substrate surface comprises a plurality of lipid coated particlesimmobilized on the substrate surface, wherein the plurality of lipidcoated particles comprises at least one functional molecule, wherein theat least one functional molecule induces coagulation; and detecting oneor more coagulation products associated with the at least one functionalmolecule of the plurality of the lipid coated particles. In one aspect,the blood product is selected from whole blood, plasma, platelet richplasma, and platelet poor plasma. In another aspect, the flow conditionssimulate hemodynamic conditions of the individual. In still anotheraspect of the method, the functional molecule is one or moretransmembrane proteins. The transmembrane proteins can be selected fromtissue factor, thromobomodulin, endothelial cell protein C receptor,glycoprotein IIb/IIIa, glycoprotein VI, glycoprotein Ib/IX/V,P-selectin, glycoprotein IV, CD9, platelet endothelial cell adhesionmolecule (PECAM-1), Ras-related protein 1b (rap1b), c-type lectin-likereceptor 2 (CLEC-2), intracellular adhesion molecule 1 (ICAM-1),intracellular adhesion molecule 2 (ICAM-2) and combinations thereof. Inyet another aspect, the functional molecule initiates coagulation. Instill another aspect, the functional molecule inhibits coagulation.

In yet another aspect of the method, the step of detecting comprisesquantifying the coagulation product. The coagulation product can beselected from thrombin, fibrin, thrombin-antithrombin complex,fibrinopeptide A, fibrinopeptide B, D-dimer, prothrombin fragment 1+2,activated factor X, activated factor V, activated factor VIIIa,activated factor IXa, activated factor XIa, activated factor XIIa,activated protein C, activated protein S, and mixtures thereof. In stillanother aspect of the method, the coagulation product can be detected bya method selected from brightfield microscopy, darkfield microscopy,fluorescence microscopy, multi-photon excitation, second harmonicgeneration, third harmonic generation, atomic force microscopy, scanningelectron microscopy, and absorbance.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts a top view of an exemplary microfluidic device with atleast some embodiments of the present invention.

FIG. 2 depicts an exploded top view of a portion of an exemplarymicrofluidic device with at least some embodiments of the presentinvention.

FIG. 3 depicts an exploded side-view of a portion of an exemplarymicrofluidic device with at least some embodiments of the presentinvention.

FIG. 4 depicts the formation of lipid coated particles of the presentinvention and patterning on glass slides. Tissue Factor (TF);L-α-phosphatidylcholine (PC) and L-α-phosphatidylserine (PS); DHPE(Texas red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine);3-[(2-Aminoethylamino) propyl] trimethoxysilane (APTMS);Polydimethylsiloxane (PDMS).

FIG. 5A depicts 100 μM patterned Tissue Factor lipid coated particles. Apluarity of lipid coated particles is found within each spot.

FIG. 5B depicts a microfluidic device with hydrodynamic focusing whichis used to force Alexa 488-labelled plasma (lightest shaded regionindicated as the “Blood Product Area”) to flow in the center over thepatterned lipid coated particles in the spots, while bounded on the sideby Texas labeled buffer (mid-shaded regions adjacent to the lightestshaded regions indicated as the “Buffer Area”).

FIG. 5C depicts fibrin that is formed over the lipid coated particlespots of FIG. 5B over a 10 minute perfusion of plasma at 0 s, whichrepresents start of perfusion.

FIG. 5D depicts fibrin that is formed over the lipid coated particlespots of FIG. 5B over a 10 minute perfusion of plasma at 300 s, whichrepresents 300 seconds after the start of perfusion.

FIG. 5E depicts fibrin that is formed over the lipid coated particlespots of FIG. 5B over a 10 minute perfusion of plasma at 600 s, whichrepresents 600 seconds (or 10 minutes) after the start of perfusion.

FIG. 5F depicts thrombin generation over the lipid coated particle spotsof FIG. 5B as measured by a blue signal that is emitted when thethrombin substrate boc-VPR-AMC is cleaved over a 10 minute perfusion ofplasma at 0 s, which represents start of perfusion.

FIG. 5G depicts thrombin generation over the lipid coated particle spotsof FIG. 5B as measured by a blue signal that is emitted when thethrombin substrate boc-VPR-AMC is cleaved over a 10 minute perfusion ofplasma at 300 s, which represents 300 seconds after the start ofperfusion.

FIG. 5H depicts thrombin generation over the lipid coated particle spotsof FIG. 5B as measured by a blue signal that is emitted when thethrombin substrate boc-VPR-AMC is cleaved over a 10 minute perfusion ofplasma at 600 s, which represents 600 seconds (or 10 minutes) after thestart of perfusion.

FIG. 6A depicts fibrin generation after normal pooled plasma (NPP) wasperfused over TF lipid coated particles using the microfluidic device ofthe present invention, wherein the TF concentration was 50 molecules/μm²at wall shear rates of 50, 100, 250, 500 and 1000 s⁻¹. RelativeFluorescence Units (RFUs) were determined in real-time using threemetrics to quantify the dynamics of fibrin generation (i) the lag timeto fibrin fiber generation, (ii) the maximum fibrin density, and (iii)the rate of fibrin generation.

FIG. 6B depicts fibrin generation after normal pooled plasma (NPP) wasperfused over TF lipid coated particles using the microfluidic device ofthe present invention, wherein the TF concentration was 5 molecules/μm²at wall shear rates of 50, 100, 250, 500 and 1000 s⁻¹. RelativeFluorescence Units (RFUs) were determined in real-time using threemetrics to quantify the dynamics of fibrin generation (i) the lag timeto fibrin fiber generation, (ii) the maximum fibrin density, and (iii)the rate of fibrin generation.

FIG. 6C depicts fibrin generation after normal pooled plasma (NPP) wasperfused over TF lipid coated particles using the microfluidic device ofthe present invention, wherein the TF concentration was 0.5molecules/μm² at wall shear rates of 50, 100, 250, 500 and 1000 s⁻¹.Relative Fluorescence Units (RFUs) were determined in real-time usingthree metrics to quantify the dynamics of fibrin generation (i) the lagtime to fibrin fiber generation, (ii) the maximum fibrin density, and(iii) the rate of fibrin generation.

FIG. 6D depicts thrombin generation after normal pooled plasma (NPP) wasperfused over TF lipid coated particles using the microfluidic device ofthe present invention, wherein the TF concentration was 50 molecules/μm²at wall shear rates of 50, 100, 250, 500 and 1000 s⁻¹. RelativeFluorescence Units (RFUs) were determined in real-time using threemetrics to quantify the dynamics of thrombin generation (i) the lag timeto thrombin generation, (ii) the maximum thrombin fluorescence, and(iii) the rate of thrombin generation.

FIG. 6E depicts thrombin generation after normal pooled plasma (NPP) wasperfused over TF lipid coated particles using the microfluidic device ofthe present invention, wherein the TF concentration was 5 molecules/μm²at wall shear rates of 50, 100, 250, 500 and 1000 s⁻¹. RelativeFluorescence Units (RFUs) were determined in real-time using threemetrics to quantify the dynamics of thrombin generation (i) the lag timeto thrombin generation, (ii) the maximum thrombin fluorescence, and(iii) the rate of thrombin generation.

FIG. 6F depicts thrombin generation after normal pooled plasma (NPP) wasperfused over TF lipid coated particles using the microfluidic device ofthe present invention, wherein the TF concentration was 0.5molecules/μm² at wall shear rates of 50, 100, 250, 500 and 1000 s⁻¹.Relative Fluorescence Units (RFUs) were determined in real-time usingthree metrics to quantify the dynamics of thrombin generation (i) thelag time to thrombin generation, (ii) the maximum thrombin fluorescence,and (iii) the rate of thrombin generation.

FIG. 7 shows the results of a D-dimer analysis of the cumulative fibrindeposited (generated) as described in FIGS. 6A-6C on all the spotscomprising a plurality of the TF lipid coated particles as measured byD-dimer concentration following plasmin digestion. The wall shear ratesare provided at the top of the graph.

FIG. 8A shows the fiber height of the fibrin fibers that accumulated onthe individual spots from the assay described in FIGS. 6A-6C wherein thefiber height is provided for each of the wall shear rates of 50, 100,250, 500 or 1000 s⁻¹ to show the shear rate effects with distribution ofthe fibrin fibers.

FIG. 8B shows final fluorescence images showing a decrease in fibrinfiber density and intensity with an increase in shear rate (scalebars=20 um) at 50 s⁻¹ from the assay described in FIGS. 6A-6C.

FIG. 8C shows final fluorescence images showing a decrease in fibrinfiber density and intensity with an increase in shear rate (scalebars=20 um) at 100 s⁻¹ from the assay described in FIGS. 6A-6C.

FIG. 8D shows final fluorescence images showing a decrease in fibrinfiber density and intensity with an increase in shear rate (scalebars=20 um) at 250 s⁻¹ from the assay described in FIGS. 6A-6C.

FIG. 8E shows final fluorescence images showing a decrease in fibrinfiber density and intensity with an increase in shear rate (scalebars=20 um) at 500 s⁻¹ from the assay described in FIGS. 6A-6C.

FIG. 8F shows final fluorescence images showing a decrease in fibrinfiber density and intensity with an increase in shear rate (scalebars=20 um) at 1000 s⁻¹ from the assay described in FIGS. 6A-6C.

FIG. 8G shows scanning electron micrographs of fibrin diameterdecreasing with an increase in shear rate at 50 s⁻¹ from the assaydescribed in FIGS. 6A-6C.

FIG. 8H shows scanning electron micrographs of fibrin diameterdecreasing with an increase in shear rate at 100 s⁻¹ from the assaydescribed in FIGS. 6A-6C.

FIG. 8I shows scanning electron micrographs of fibrin diameterdecreasing with an increase in shear rate at 250 s⁻from the assaydescribed in FIGS. 6A-6C.

FIG. 8J shows scanning electron micrographs of fibrin diameterdecreasing with an increase in shear rate at 500 s⁻¹ from the assaydescribed in FIGS. 6A-6C.

FIG. 8K shows scanning electron micrographs of fibrin diameterdecreasing with an increase in shear rate at 1000 s⁻¹ from the assaydescribed in FIGS. 6A-6C.

FIG. 9A shows fibrin generation for factor deficient plasma at 50 s⁻¹and TF concentration of 50 molecules/μm² using the assay described inFIGS. 6A-6C. Normal pooled plasma (NPP); plasma deficient with factor XI(FXI-def); plasma deficient with factor VIII (FVIII-def); and plasmadeficient with factor IX (FIX-def).

FIG. 9B shows fibrin generation for factor deficient plasma at 5 s⁻¹ andTF concentration of 5 molecules/μm² using the assay described in FIGS.6A-6C. Normal pooled plasma (NPP); plasma deficient with factor XI(FXI-def); plasma deficient with factor VIII (FVIII-def); and plasmadeficient with factor IX (FIX-def).

FIG. 9C shows thrombin generation for factor deficient plasma at 50 s⁻¹and TF concerntration of 50 molecules/μm² using the assay described inFIGS. 6D-6F. Normal pooled plasma (NPP); plasma deficient with factor XI(FXI-def); plasma deficient with factor VIII (FVIII-def); and plasmadeficient with factor IX (FIX-def).

FIG. 9D shows thrombin generation for factor deficient plasma at 5 s⁻¹and TF concentration of 5 molecules/μm² using the assay described inFIGS. 6D-6F. Normal pooled plasma (NPP); plasma deficient with factor XI(FXI-def); plasma deficient with factor VIII (FVIII-def); and plasmadeficient with factor IX (FIX-def).

DETAILED DESCRIPTION

This invention generally relates to a microfluidic device and methodsand uses of the device for evaluating and testing a blood product froman individual as well as for measuring the clotting characteristics of ablood product from an individual. This invention describes a flow basedassay that allows the use of a blood product such as plasma formeasuring end products of the coagulation cascade (such as thrombin andfibrin generation). The advantage of this invention is that itintegrates the coagulation cascade into a fluidic architecture, whichallows for measurement of coagulation products under the hemodynamicconditions found in the body. Furthermore, because this assay allows theuse of plasma, the plasma samples can be stored for long periods of timebefore being tested. This is in contrast to most flow based assays thatuse only whole blood, which needs to be used within hours of a blooddraw.

This invention fills a technology gap for a flow based plasma assay formeasuring coagulation potential.

There are no known flow based plasma assays for coagulation. Staticplasma assays for measuring coagulation include thrombin generation(TG), prothrombin time (PT), partial thromboplastin time (PTT), andturbidity-based assays.

With reference to FIG. 1 an embodiment of the present invention isillustrated. This is an exemplary microfluidic device in accordance withat least some embodiments of the present invention. More specifically,the microfluidic device may include one or more fluid receiving passageswhich allows for fluid to flow through a microfluidic device; an inletfor a blood product (such as plasma) which is capable of receiving ablood product (shown as “2”) and an outlet (shown as “3”) where bloodproduct can be collected. The microfluidic device may also include abuffer inlet for hydrodynamic focusing (shown as “1”) which is capableof receiving buffer. Each shaded circular spot represents a plurality oflipid coated particles of the invention that is attached to a substratesurface. The arrows represent the direction of flow of the blood productand buffer through the channels of the microfluidic device.

With reference to FIG. 2 an embodiment of the present invention isillustrated. This is an exploded top view of an exemplary microfluidicdevice as described in FIG. 1 with at least some embodiments of thepresent invention. Each shaded circular spot represents a plurality oflipid coated particles of the invention that plasma flows over. Thebuffer sections represent buffer that is flowed adjacent to the plasmaover the spots demonstrating focusing of the plasma on the spots.

With reference to FIG. 3 an embodiment of the present invention isillustrated. This is an exploded side-view of a portion of an exemplarymicrofluidic device as described in FIG. 1 with at least someembodiments of the present invention. Each circle represents anindividual single lipid coated particle within a single circular spot asdepicted in FIGS. 1 and 2.

The device of the present invention comprises at least one microfluidicchannel. The microfluidic device may include a plurality of fluid-filledreceiving passages, which are capable of receiving fluid at a receivingend and allowing the fluid to flow through a microfluidic channel to acollection point or terminal end. One or more microfluidic channels maybe present and may spilt into multiple channels, thereby resulting in anumber of terminal ends. The number of receiving ends may equal thenumber of terminal ends. The configuration and design of themicrofluidic channels can vary without departing from the scope of thepresent invention.

In addition to comprising at least one microfluidic channel, themicrofluidic device may also comprise at least one substrate surfacewhich intersects one or more of the microfluidic channels. In addition,the substrate surface can be functionalized. In this step, the substratesurface may be treated with 3-aminopropyl-trimethoxysilane (APTMS),thereby creating a monolayer of APTMS on the upper surface of thesubstrate. Methods of rendering substrates, such as glass substrates,hydrophilic are well known in the art. Method of functionalizing thesubstrate include, without limitation, rendering the substrate and/orthe substrate surface hydrophilic, hydrophobic, reactive (via amine orcarboxylic acid groups) or some other chemistry. In one embodiment,silane chemistries may be used on the substrates. The substrate may beany composed of any material including but not limited to glass,plastic, gold, quartz, silicon, silicon nitride, silicon dioxide,polydimethylsiloxane, polystyrene, polymethyl methacrylate andcombinations thereof, or any other type of known substrate material usedin surface chemistry. Additionally, the substrate is a size that allowsfor complete immersion of the substrate and/or substrate surface intothe microfluidic channel.

The substrate surface comprises a plurality of lipid coated particlesthat are immobilized on the substrate surface. The lipid coatedparticles may be comprised of silica such as silica glass or ceramics,including but not limited to silica beads that are synthesized bymethods known to those of skill in the art, including but not limited tothe Stober process. The resulting silica beads may be silica micro beadsand may range in diameter from about 0.1 micrometer to about 100micrometers. Preferably, the resulting silica beads are 1 to 10micrometers in diameter.

In the case of lipid coated particles formed using silica beads, oncethe silica beads are synthesized they may be made hydrophilic by usingknown methods including but not limited to treatment with hydrogenperoxide and dilute organic acid. Once the beads are synthesized andmade hydrophilic, their surfaces may be coated with lipid bilayerscomprised of one or more phospholipid structures. These phospholipidstructures include but are not limited to phosphatidylserine,phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine,phophoinositides, phosphosphingolipids, and combinations thereof. Thecomposition of the lipid bilayer may be altered to mimic the surface ofvarious cell types, such as platelets, leukocytes, erythrocytes,endothelial cells or smooth muscle cells. This alteration may beaccomplished by mixing different ratios of the phospholipid structures,such as phosphatidylserine and phosphatidylcholine andphosphatidylethanolamine. Other combinations of two or more of thephospholipid structures may also be mixed.

The lipid coated particles in addition to being coated with lipidbilayers, also are comprised of one or more functional molecules whichare contained within the lipid bilayers. The functional molecule may beany transmembrane protein. Such transmembrane protein may includetransmembrane proteins that are known to regulate blood coagulationincluding but not limited to tissue factor, thromobomodulin, endothelialcell protein C receptor and combinations thereof. Thrombin is a knownserine protease that creates a biopolymer of fibrin by cleavingfibrinopeptide from the plasma protein of fibrinogen. Fibrin forms ahighly entangled hydrogel that provides the scaffold onto which a bloodclot grows. Generally, high concentrations of thrombin are createdduring the extrinsic or tissue factor pathway of the coagulationcascade, hence why tissue factor is known as a coagulation cascadeinducing agent. Other transmembrane proteins may include proteins thatare known to be receptors for cell to cell adhesion including but notlimited to glycoprotein IIb/IIIa, glycoprotein VI, glycoprotein Ib/IX/V,P-selectin, glycoprotein IV, CD9, platelet endothelial cell adhesionmolecule (PECAM-1), Ras-related protein 1b (rap1b), c-type lectin-likereceptor 2 (CLEC-2), intracellular adhesion molecule 1 (ICAM-1),intracellular adhesion molecule 2 (ICAM-2) and combinations thereof. Thelipid coated particles may contain various concentrations of one or moreof the functional molecules.

Once the lipid coated particles are synthesized and coated as discussedabove, they are immobilized to the substrate surface. Methods toimmobilize silica beads, such as the lipid coated particles of thepresent invention, are known to those of skill in the art and includebut are not limited to covalent bonding, electrostatic interactions andhydrogen bonding. The immobilization method provides an adequateattractive force between the lipid coated particles and the substratesurface to withstand shear stresses during the assay.

In a further aspect, the lipid coated particles are immobilized andpatterned to the substrate surface. The patterning may be achieved by asubtractive technique such as microblotting or a lift-off process suchas microstenciling. In regards to microblotting, a blotting device maybe loaded onto a mechanical press and lowered onto the substrate surfacecomprising a plurality of the lipid coated particles until the blottingdevice is in full contact with the substrate surface comprising theimmobilized lipid coated particles. Once the blotting device is removed,defined sections and/or areas (i.e. the resulting pattern) of theimmobilized lipid coated particles remain on the substrate surface (seeFIG. 4). The defined sections and/or areas may be of any geometric shapeincluding but not limited to circular shape, square shape, oval shape,rectangular shape or unshaped and combinations thereof. A circular shapemay be referred to as a “spot”. Each defined section and/or area iscomprised of a plurality of the lipid coated particles. In each definedsection and/or area, there is at least more than one lipid coatedparticle. Preferably, there are hundreds to thousands of lipid coatedparticles present in each defined section and/or area. The number ofindividual lipid coated particles within each defined section and/orarea can vary and can be in a range from 1 to about 1,000,000 individuallipid coated particles. It is possible for the defined section and/orarea to be comprised of a single lipid coated particle. As used herein aplurality of lipid coated particles refers to at least more than onelipid coated particle. The diameter of each defined section and/or areacan vary as determined by the patterning method. One or more definedsections and/or areas may result depending on the patterning method. Atleast one defined section and/or area comprising a plurality of thelipid coated particles results depending on the pattern. The number ofdefined sections and/or areas found on the substrate surface can vary asdetermined by the patterning method, the composition of the lipidbilayer and the measurement being taken. The number of defined sectionsand/or areas can be from about 1 to 1000 defined sections and/or areas.The spacing between two or more defined sections and/or areas of theplurality of the lipid coated particles on the substrate surface mayvary depending on the pattern. As an example, the space between two ofthe defined sections and/or areas may be about 1 μm apart to about 1 mmapart. Each lipid coated particle within the defined section and/or areais smaller than the diameter of the microfluidic channel itself.

The width of the substrate surface comprising the immobilized lipidcoated particles may vary depending upon the size of the microfluidicchannel. In some embodiments the width may be about 10 micrometers (μm),about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 100 μm, about150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about400 μm, about 450 μm, about 500 μm, about 600 μm, about 700 μm, about800 μm, about 900 μm or about 1000 μm. The actual width of the substratesurface can have a greater or lesser size without departing from thescope of the present invention.

After the plurality of the lipid coated particles are immobilized andpatterned, the particles are integrated into at least one microfluidicchannel by aligning either manually, such as by using alignment marksmade on the substrate surface, or having posts on the substrate thatalign with holes that can be found on the microfluidic device or byother methods known to those of skill in the art. Once aligned, thesubstrate and the device are bonded. Such bonding method includes but isnot limited to vacuum assisted bonding.

In a further embodiment, the microfluidic device has the capability ofhydrodynamic focusing. Additional buffer, such as HEPES buffered saline(HBS) may be infused through additional side channels of the device toprovide focusing of the sample, such as a sample of a blood product,which is perfused through a middle channel. As the buffer solution isperfused in from the side it forces the sample to flow in the middlepart of the channel. This design prevents edge effects, most notably,the accumulation of the deposition in the corners of the channel of thesample and/or sample product (see FIG. 5B).

Once the device is produced, in accordance with at least someembodiments of the present invention, the microfluidic device may behygienically sealed in a sterile environment (e.g. hermetic plasticpackage) such that the microfluidic device can be distributed as a clottesting kit to medical personnel and other interested parties. Inaddition, the substrate surface comprising the lipid coated particlescan be kept in an aqueous environment. Accordingly, prior tohermetically sealing the microfluidic device in a sterile environment,an aqueous solution may be injected into the hermetic packaging prior tothe final sealing. Alternatively, the substrate surface may be kept in adry environment.

Another embodiment of the invention is a microfluidic channel throughwhich a blood product is capable of flowing. The channel may comprise atleast one substrate provided as part of at least a portion of onesurface of the channel. The substrate surface comprises a plurality ofthe lipid coated particles of the invention. These particles comprise atleast one functional molecule that is embedded in the lipid coating theparticle. The functional molecule is as defined herein. As used hereinthe term blood product refers to whole blood, plasma, platelet richplasma (defined as having no red or white blood cells, while containingplasma and platelets), and platelet poor plasma (defined as having noplatelets). The blood product of the present invention may be from anindividual, such as whole blood or plasma taken from an individual orthe blood product may be synthetically produced by methods known in theart.

In accordance with at least some embodiments of the present invention,once the microfluidic device has been prepared, one or more bloodcomponent samples can be passed or perfused through the microfluidicchannels of the device under flow conditions to evaluate the bloodproduct for coagulation products associated with at least one of thefunctional molecules of the coated lipid particles. As noted previously,the functional molecules may be one or more transmembrane proteinsincluding but not limited to tissue factor, thromobomodulin, endothelialcell protein C receptor, glycoprotein IIb/IIIa, glycoprotein VI,glycoprotein Ib/IX/V, P-selectin, glycoprotein IV, CD9, plateletendothelial cell adhesion molecule (PECAM-1), Ras-related protein 1b(rap1b), c-type lectin-like receptor 2 (CLEC-2), intracellular adhesionmolecule 1 (ICAM-1), intracellular adhesion molecule 2 (ICAM-2) andcombinations thereof. In a preferred embodiment, the functional moleculeinitiates coagulation, such a tissue factor. In another embodiment, thefunctional molecule inhibits coagulation, such as thrombomodulin.

The flow conditions and rate can be defined by the user of the device.Preferably, the flow conditions simulate hemodynamic conditions of anindividual for which the blood product is obtained from. The flowconditions may include a wall shear rate of in the range of about 50sec⁻¹ to about 2600 sec⁻¹, which corresponds to the normal range shearrates in the human vasculature. The flow conditions may also includewall shear rates in a range from zero up to about 500,000 sec⁻¹ fortesting conditions in which the pathological flow conditions exist.Pathological flow conditions may occur if the flow has been retarded, asin the case of individuals diagnosed deep vein thrombosis, or if bloodis forced to pass through a partially occluded or stenosed vessel, as inthe case of individuals diagnosed with atherosclerosis.

As the one or more blood component samples are perfused through themicrofluidic channel and over the substrate surface comprising aplurality of the lipid coated particles comprising one or morefunctional molecules of the present invention, and the sample isperfused at a user defined flow rate, coagulation products can bedetected as one or more coagulation products associates with the coatedlipid particles comprising the functional molecule. Coagulation productsinclude but are not limited to thrombin, fibrin, thrombin-antithrombincomplex, fibrinopeptide A, fibrinopeptide B, D-dimer, prothrombinfragment 1+2, activated factor X, activated factor V, activated factorVIIIa, activated factor IXa, activated factor XIa, activated factorXIIa, activated protein C, activated protein S, and mixtures thereof.The coagulation products may be detected and/or measured by variousmethods including but not limited to brightfield microscopy, darkfieldmicroscopy, fluorescence microscopy, multi-photon excitation, secondharmonic generation, third harmonic generation, atomic force microscopy,scanning electron microscopy, and absorbance. For example, thrombinand/or fibrin amounts can be detected and/or measured by using afluorescent substrate such as boc-VPR-AMC for thrombin and for fibrin byadding exogenous fibrinogen with a fluorescent label. Thrombingeneration could also be indirectly detected and/or measured bymeasuring collecting the effluent at the outlet of the device andmeasuring the concentration of thrombin-antithrombin complex (TAT) orthe release of fibrinopeptides (peptides that are released off offibrinogen following cleavage by thrombin). Fibrin could also bedetected and/or measured using a fibrin specific antibody or by othermicroscopy techniques such as differential contrast, phase contrast andHoffman modulation. Any of the other transmembrane protein could bedetected and/or measured in similar ways to those described above. Anindividual's result may be compared to results that have been obtainedunder identical conditions using plasma pooled from normal donors (i.e.normal pooled plasma (NPP) which is plasma that has been pooled from anumber of normal donors (donors without known blood coagulationconditions)). The NPP can be plasma standards that are availablecommercially. Additionally, an individual's results can be compared toresults that have been obtain under identical conditions using factordeficient plasmas such as factor II (prothrombin), factor VIII, factorIX, factor and factor XI deficient plasmas.

Another embodiment of the present invention relates to a method fordetermining an individual's coagulation potential, comprising perfusingan individual's blood product (such as plasma) over the substratesurface comprising the coated lipid particles of the present inventionand one or more functional molecules, wherein one or more coagulationproducts associate with the lipid particles; and detecting one or morecoagulation products associated with the lipid particles. In one aspect,the lipid particles comprise functional molecules. The functionalmolecules can initiate coagulation or can inhibit coagulation. In apreferred aspect, the functional molecules are tissue factor andthrombomodulin. The flow rate of the blood product can be a rate whichmimics hemodynamic conditions of the individual. The normal range ofshear rates in the human vasculature is about 50 to 2600 sec⁻¹.

Another embodiment of the invention relates to a method for determiningan individual's response to an agent comprising perfusing theindividual's blood product (such as plasma) over the substrate surfacecomprising the coated lipid particles of the present invention and oneor more functional molecules wherein one or more coagulation productsassociate with the lipid particles; and detecting one or morecoagulation products associated with the lipid particles. In one aspect,the agent is an anticoagulant agent or coagulating agent.

In still another embodiment, the invention relates to a method todiagnose and/or monitor an individual for bleeding comprising perfusingthe individual's blood product (such as plasma), over the substratesurface comprising the coated lipid particles of the present inventionand one or more functional molecules wherein one or more coagulationproducts associate with the lipid particles; and detecting one or morecoagulation products associated with the lipid particles.

In yet another embodiment, the invention relates to a method todetermine the dose of one or more anticoagulation agents or coagulationagents to be administered to an individual comprising perfusing theindividual's blood product (such as plasma), over the substrate surfacecomprising the coated lipid particles of the present invention and oneor more functional molecules, wherein one or more coagulation productsassociate with the lipid particles; and detecting one or morecoagulation products associated with the lipid particles.

Yet another embodiment relates to a method to screen for anticoagulationagents or coagulation agents comprising perfusing the individual's bloodproduct (such as plasma), over the substrate surface comprising thecoated lipid particles of the present invention and one or morefunctional molecules, wherein one or more coagulation products associatewith the lipid particles; and detecting one or more coagulation productsassociated with the lipid particles.

Still another embodiment relates to a method to screen for coagulationagents comprising perfusing the individual's blood product (such asplasma), over the substrate surface comprising the coated lipidparticles of the present invention and one or more functional molecules,wherein one or more coagulation products associate with the lipidparticles; and detecting one or more coagulation products associatedwith the lipid particles.

The individual in the invention can include any mammal, including humanand non-human mammals.

The following experimental results are provided for purposes ofillustration and are not intended to limit the scope of the invention.

EXAMPLES Example 1

This example demonstrates the fabrication of lipid coated particles ofthe present invention to detect fibrin formation.

In order to initiate fibrin formation in the plasma-based model forcoagulation using the microfluidic device of the present invention,silica microbeads are used to simulate the catalytic activity ofplatelets. Once the silica beads are made hydrophilic, their surfacesare coated with lipid bilayers, which contain varied concentrations offunctional molecules such as tissue factor (TF) and thrombomodulin (TM)that initiate and inhibit coagulation. The composition of the lipidbilayer can be manipulated to mimic the surface of various cell types.This is accomplished by mixing different ratios of phospholipids such asphosphatidylserine (PS), phosphatidylcholine (PS), andphosphatidylethanolamine (PE). A description of the fabrication ofsilica beads and coating them with phospholipids is found below:

1. Weigh or measure out 2.1 mL of deionized water, 15.4 mL of anhydrousethanol, and 6.5 mL of ammonium hydroxide in a plastic container.

2. Stir for 10 minutes with a magnetic stirrer, and then add 1.5 mLtetraethyl orthosilicate (TEOS) dropwise.

3. Stir the solution for 2 hours at the room temperature.

4. After 2 hours, centrifuge the precipitated silica beads, and wash inethanol 4 times.

5. Resuspend the bead solution of 400 mg in 1 ml of deionized water.

6. Dilute the silica particles to a concentration of 5 mg/ml in DI water

7. Add hydrogen peroxide and HCl solution to the mixture to finalconcentrations of 4% vol and 0.4 M, respectively.

8. Stir the solution at 85° C. for 10 minutes, then cool the mixture toroom temperature.

9. Wash the solution of beads by centrifuging the beads at 2000 RPM for5 minutes and re-suspending in buffered saline to wash the beads. Repeat5 times

10. Pipette 200-500 uL of the silica bead in a microcentrifuge tube andcentrifuge the silica beads at 2000 RPM for 3 minutes.

11. Pipette out the supernatant buffer without drying out the silicabeads and add 100 uL of liposome .Vortex the mixture until the silicabeads are re-suspended in the lipid solution

12. Incubate the lipid-bead mixture for 24 hours at 4° C. to allow thelipid bilayers to rupture on the silica bead surfaces

13. Centrifuge the silica beads at 2000 RPM for 5 minutes, then pipetteoff the supernatant solution. Do not dry out the beads.

14. Rinse the beads thoroughly with a 1× HBS buffer

15. Re-suspend the lipid coated silica beads to concentration of 1 mg/mLand store at 4° C. until ready to use.

Example 2

This example demonstrates an example of the patterning of lipid coatedparticles of the present invention.

Once the lipid coated particles are made as described in Example 1, theyneed to be immobilized and patterned onto a substrate. Theimmobilization of the particles can rely on methods such as covalentbonds (e.g. streptavidin-biotin), electrostatic interactions, orhydrogen bonding. The patterning may be achieved either by a subtractivetechnique such as microblotting or a lift-off process such as amicrostencil. A microblotting technique is described below:

1. Obtain a 100 μg/mL solution of silica beads functionalized with lipidbilayer as explained in Example 1.

2. Incubate a 100 μg/mL solution of the lipid coated particles in HBSbuffer onto and APTES functionalized glass slide for 1 hour at roomtemperature. Wash substrates three times in HBS buffer to remove unboundbeads.

3. Place glass slide into a 6-inch petri dish with 100 mL of deionized18 MΩ water.

4. Load blotting devices onto a customized mechanical press and loweronto the slide until the device is in full contact with the slide. Thedevice is contacted on the slide for 3 minutes.

5. Thoroughly rinse the patterned slide in PBS or HBS for 5 min. Storethe patterned slide in buffer until use in the flow assay.

Example 3

This example demonstrates an example of the integration of the lipidcoated particles into microfluidic channels of the present invention.

After immobilizing and/or patterning the lipid coated particles, themicrofluidic device of the present invention is aligned and bonded tothe substrate. Because the lipid bilayers on the lipid coated particlesshould remain hydrated, this step should be done while the substrate isimmersed or covered in buffer solution. The device may be aligned eithermanually or using alignment marks on the substrate. Once in contact, thedevice may be bonded to the substrate by vacuum assisted bonding.

Example 4

The example demonstrates how to measure coagulation under flowconditions.

The procedure for the assay itself using the microfluidic device of thepresent invention comprises perfusing plasma through the microfluidicchannels and over the plurality of the lipid coated particles at a userdefined flow rate. Coagulation can be monitored by a variety of opticalmethods: (1) fibrin generation by brightfield microscopy (phasecontrast, Hoffman modulation contrast, differential interferencecontrast), (2) fibrin generation by fluorescence microscopy(epifluorescence or confocal), which requires that either somefibrinogen is labeled with a fluorophore or the inclusion of afibrinogen or fibrin fluorescence labeled antibody into the plasma, (3)fibrin generation by nonlinear optical methods (two photon excitation,second harmonic generation, third harmonic generation), (4) thrombingeneration by monitoring the fluorescence or absorbance of a thrombinsubstrate (e.g. boc-VPR-AMC), (5) thrombin generation by measuringthrombin-antithrombin (TAT) complex, (6) fibrin deposition bymeasurement of the D-dimer following digestion by plasmin, (7) fibringeneration by measurement of fibrinopeptides A and B.

Example 5

This example demonstrates the use of the microfluidic device of thepresent invention to measure transient fibrin deposition and thrombingeneration. The Inventors demonstrate that for a given Tissue Factor(TF) concentration, flow profoundly influenced fibrin deposition, fiberdiameter, fiber orientation and local thrombin concentration. Themicrofluidic device can also be used to investigate the effects ofdifferent factor deficiencies on the dynamics of fibrin production andthrombin generation.

These findings suggest that for significant fibrin formation to occur,coagulation reactants and products must be protected from transport awayfrom a clot either by a reduction in shear rate (i.e. occlusion orwithin secondary flows downstream of stenosis) or within theinterstitial spaces of a platelet aggregate.

Preparation of TF Lipid Coated Particles and Assay Conditions Using theMicrofluidic Device

TF bearing lipid coated particles were synthesized by coating 1 μmsilica beads with 0.5, 5, or 50 molecules TF/μm² in a lipid bilayer(PS:PC 30:70). The lipid coated particles were patterned as 100 μm spotson a glass substrate using a microblotting technique. (FIG. 5A). Normalpooled plasma (NPP), factor deficient plasmas FII, FVIII, FX and FXIwere perfused over the TF spots at wall shear rates of 50, 100, 250, 500and 1000 sec⁻¹ for 10 min. (FIG. 5B). Fibrin formation and thrombingeneration were measured in real-time by epifluorescence using labeledfibrinogen and the thrombin substrate boc-VPR-AMC, respectively. (FIGS.5C-5H). Following the assay, fibrin gels were either (i) fixed andfurther imaged by confocal or scanning electron microscopy or (ii)digested by plasmin to measure the rate of lysis and to quantify theamount of fibrin deposited using a D-dimer ELISA.

Shear Rate Dependent Fibrin Deposition and Thrombin Generation

NPP was perfused over surface TF concentration of 0.5, 5 and 50molecules/μm² at wall shear rates of 50, 100, 250, 500 and 1000 sec⁻¹.For each experiment, fibrin deposition and thrombin generation weremonitored in real-time. Fibrin deposition and thrombin generationdecreased with increasing wall shear rate and decreasing TFconcentration. (FIGS. 6A-6F). Three metrics were used to quantify thedynamics of fibrin formation and thrombin generation; (i) the lag timeto fibrin fiber and thrombin generation, (ii) the maximum fibrin densityand thrombin fluorescence, and (iii) the rate of fibrin and thrombingeneration.

At a given TF concentration either of 50, 5 and 0.5 molecules/μm² therewas a decrease in the lag time and fibrin generation rate withincreasing wall shear rate. As a result of the decrease in the rate offibrin production, the maximum fibrin deposited also decreased with anincrease in shear rate. The thrombin generation followed the same trendas the fibrin deposition.

At the highest shear rates and lowest tissue factor concentration, nofibrin fibers were observed in the time period of the experimentstherefore there was a subthreshold amount thrombin produced to inducefibrin formation.

The cumulative fibrin deposited on all spots over the 10 minute flowassay was measured by D-dimer concentration following plasmin digestion.The threshold nature of fibrin formation is evident at all three TFconcentrations. For 5 and 50 molecules/μm², fibrin formation wassupported at wall shear rates less than or equal to 250 s⁻¹. For 0.5molecules/μm², fibrin formation was supported at wall shear rates ofless than or equal to 100 s⁻¹.

After plasma perfusion, the fibrin fibers produced on the TF spots weredigested with plasmin over time and monitored by the decrease influorescence intensity over time. The analysis of the rate of digestionshows that the fibers produced at the lowest shear rate digested thefastest (46.1 RFU/s) (i.e. relative fluorescence units/second), whilethe fibers produced at the highest shear rate digested the slowest (17.3RFU/s). Overall, the D-dimer analysis shows a decrease in the quantityof D-dimer fragments as shear rate is increased and tissue factorconcentration is decreased (FIG. 7). The D-Dimer results also confirmthat at the lowest TF concentration and the highest shear rates therewas little or no fibrin observed to be produced because the signals werethe same as that of the ELISA background.

Cross-Talk Between Spots

In each assay there were 142±11 fibrin(ogen) lipid coated particle spotswith a spot-to-spot distance of 200 μm. There was an increase inaccumulation of fibrin from upstream to downstream spots at shear ratesof 50 s⁻¹ and 100 s⁻¹. There were also vertical spot to spotinteractions at all shear rates where fibrin was produced, depending onthe distance from the leading spot upstream. Fibrin monomers were beingtransported downstream with flow. This trend was evident at all shearrates where fibrin was produced.

Shear Rate Effects on Fibrin Morphology

The final gel-height of the fibers accumulated on the individual spotsalso showed the same trend as the fibrin deposition (FIG. 8A). Thelowest shear rate had a height of 15.3 μm, while the highest shear ratehad a height of 2.1 μm. Fibrin fibers align in the direction of flow ina shear rate dependent manner (FIGS. 8B-8F). At 50 s⁻¹ and 100 s⁻¹, thefibers were isotropically oriented in a starburst pattern. Withincreasing wall shear rates of 250 s⁻¹ to 1000 s⁻¹, the fibers becomemore orientated with flow. Fibrin fiber diameter also decreases withincreasing wall shear rate (FIGS. 8G-8K). The lowest shear rate had thelargest diameters, with individual fibers appearing to be composed ofsmaller fibers joint together to form bigger ones. At the highest shearrate, the spots were scanned and no discernable fibers could be foundunder the scanning conditions used.

The Effect of FVIII, FIX, and FXI Deficiencies on Fibrin Deposition

NPP and FII, FVIII, FIX, FX and FXI deficient plasma were perfused inthe microfluidic device at the conditions of low shear rates and high TFconcentrations. As controls, FII and FX deficient plasmas showed novisible fibrin production at 50 s⁻¹ or at any other shear rates tested.At 50 molecules/μm², there was a slightly prolonged lag time for FVIII,FIX and FXI deficient plasma compared to NPP, however the final fibrindeposition was similar (FIG. 9A). The difference between NPP and thesedeficient plasmas at high TF concentrations was more evident in thethrombin generation data (FIG. 9C). FVIII deficient plasma wasindistinguishable from NPP. FIX deficient plasma had a reduced rate ofthrombin generation. FXI deficient plasma had a prolonged lag timecompared to NPP. At low TF concentration (5 molecules/μm²), there wasreduced fibrin deposition and thrombin generation for FVIII, FIX, andFXI deficient plasmas compared to NPP. There was almost a completeabsence of fibrin for FVIII and FIX deficient plasma, while the fibrindeposition for the FXI deficient plasmas was significantly reduced. Thetrends for thrombin generation were similar to fibrin deposition at lowTF (FIG. 9B and 9D).

Materials and Methods and Data Analysis for Example 5

Materials: L-α-phosphatidylcholine (PC) and L-α-phosphatidylserine (PS)were purchased from Avanti Polar Lipids (Alabaster, Ala., USA). Texasred 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE) waspurchased from Invitrogen (Carlsbad, Calif. USA). Bio-Beads SM-2 werepurchased from BioRad Laboratories (Hercules, Calif., USA). Sodiumdeoxycholate was purchased from CalBiochem (Gibbstown, N.J., USA).Boc-Val-Pro-Arg-MCA[t-Butyloxyl-carbonyl-L-Valyl-L-Prolyl-L-Arginine-4-Methyl-Coumaryl-7-Amide],was purchased from Peptide Institute Inc, Osaka, Japan and a 10 mM stocksolution was prepared according to manufacturer instruction. Recombinanthuman tissue factor (TF), IMUBIND tissue factor ELISA and D-Dimer ELISAwere from America Diagnostica (Stamford, Conn.), and were used accordingto the manufacturer's instructions. Plasmin was purchased from EnzymeResearch Laboratories (South Bend, Ind.). Normal pooled plasma (NPP) andfactor XI, X, IX, VIII, VII and II deficient plasmas were purchased fromGeorge King Biomedical (Overland Park, Kans.). Alexa Fluor 488 proteinlabeling kit (Invitrogen, Carlsbad, Calif., USA) was used to labelfibrinogen according to the manufacturers instruction.Polydimethylsiloxane (PDMS) used for microfluidic devices (Dow Corning,Sylgard 184) was purchased from Ellsworth Adhesives (Germantown, Wis.).HEPES buffered saline (HBS, 20 mM HEPES, 150 mM CaCl₂, pH 7.4) was madein house. 3-[(2-Aminoethylamino) propyl] trimethoxysilane (APTMS),tetraethyl orthosilicate (TEOS) and all other chemical were purchasedfrom Sigma Aldrich (St. Louis, Mo.).

Preparation of Lapidated Tissue Factor

Recombinant human tissue factor was incorporated into liposomesaccording to previously developed protocols (Smith and Morrissey,Journal of Thrombosis and Haemostasis, 2:1155-1162). Briefly, PC, PS andDHPE lipids were mixed at a 80:19.5:0.5 molar ratio in chloroform anddried under vacuum for 1 h. The dried film was resuspended in 1 mL of 20mM sodium deooxycholate in HBS, and allowed to hydrate for 1 h at roomtemperature. TF was then added to the lipid mixture and incubated for 10minutes (8700:1 lipid:TF). Detergent was removed from the lipid solutionwith 50 mg of Biobeads under agitation for 90 min. Next, an additional350 mg of Biobeads were added to the same solution, and agitated foranother 90 minutes. Finally, the beads were allowed to settle and thesupernatant TF was collected. The concentration of the lipidated TF wasdetermined by ELISA to be 460 nM.

Preparation of Silica Beads

The Stober process was used to synthesize silica beads used in thisstudy. Tetraethylorthosilicate (TEOS) was added drop wise to a mixtureof water, ethanol and ammonium hydroxide, and the solution was stirredfor 2 h at room temperature. The resulting solution was centrifuged at2000 rpm for 5 minutes, washed in ethanol and suspended in HBS buffer.Finally, transmission electron microscopy (TEM) was used to characterizethe size distribution of these silica particles as ranging from 800 to1000 nm.

Preparation of TF Lipid Coated Particles

To promote the formation of lipidated TF on the surface of the 1 μmsilica beads, the beads were first made hydrophilic by suspending themat a concentration of 5 mg/ml in 4% peroxide and 0.4 M HCl solution.Then, the suspension was heated to 80-90° C. for 10 min, cooled to 25°C., centrifuged at 2000 rpm for 5 min, washed three times with deionizedwater and finally re-suspended in FIBS buffer. To confirm that thesurface chemistry was successful, Fourier transform infraredspectroscopy (FTIR) was used to confirm the presence of depositedsurface silanol groups (SiOH groups) on the silica particles. Next, thedesired concentration of beads was pipetted from the stirred stocksuspension, centrifuged, and the supernatant was replaced by the desiredlipidated TF concentration. The suspension was gently vortexed for 30min. and then allowed to sit undisturbed for 5 min. Finally, the beadswere finally centrifuged and washed three times with HBS buffer toremove unbound lipids from the solution.

Microblotting TF Lipid Coated Particles

Clean glass slides were incubated in a 40 wt % APTMS in ethanol solutionfor 45 minutes. The amine group on the APTMS renders the surfacepositively charged. The negatively charged TF-coated silica beads wereincubated for 4 h on the positively charged glass slides and then rinsedwith HBS to remove excess silica beads. Next, a PDMS microblot with 100μm holes spaced 200 μm center-to-center was used selectively removebeads from the surface. Electrostatic interactions between the beads andthe surface provides an adequate attractive force to withstand the shearstresses during the flow assays.

Plasma Flow Assay

A polydimethylsiloxane (PDMS) microfluidic hydrodynamic focusing device(w=1000 μm, g=100 μm) was vacuum-sealed to the glass slide containingthe patterned lipid coated particles. Within each channel there was 5×22array of 100 μm bead spots. HBS was infused through the two sidechannels to provide the focusing of plasma, which was perfused throughthe middle channel (FIG. 5B). The total flow rate (HBS and plasma)through the main channel was set to achieve the desired wall shear rateusing the expression: γ=6×Q/H² W where γ is the shear rate, Q is thevolumetric flow rate, W is the width and H is the height. As the buffersolution was perfused in from the side (mid-shaded regions adjacent tothe lightest shaded regions indicated as the “Buffer Area”) it forcedthe plasma (lightest shaded region indicated as the “Blood ProductArea”) to flow in the middle part of the channel. This design preventsedge effects, notably the preferential accumulation of fibrin depositionin the corners of the channel.

Citrated normal pooled plasma (NPP), FII, FX, FVIII, FIX and FXIdeficient plasmas were defrosted at 37° C. immediately before perfusionthrough the microfluidic flow device. The citrated plasma (400 μL) wasre-calcified by adding 20 μL of a solution of CaCl₂ (500 mM) andwithdrawn with a syringe pump at wall shear rates of 50, 100, 250, 500and 1000 s⁻¹. To monitor fibrin formation, Alexa 488 labeled fibrinogenwas added to the plasma at 17.5 μg/mL. Thrombin generation was monitoredthrough the cleavage of a fluorogenic substrate, Boc VPR-AMC.

Data Acquisition and Image Analysis

Fibrin deposition and thrombin generation were measured for 10 minutes,and images were recorded every 50 s by epifluorescence microscopy usinga 40× objective. The data was taken starting from the leading spotupstream where the plasma first encountered the lipidated-TF lipidcoated particle bead spot pattern. Image J software was used todetermine the integrated fluorescence of the fibrin or thrombingenerated on single bead spots.

Plasmin Digestion and D-Dimer Level Measurements of Fibrin Deposits

After the plasma perfusion, the heparin wash buffer was used to rinsethe channel for 5 min. at the same shear rate as the experiment. Next, a250 μL plasmin solution (0.48 mg/ml diluted in HBS containing 1 mMTris/HCl, pH 7.4) was perfused through the microfluidic channel at aflow rate of 5 μL/min for 10 min, and then flow was stopped for 30 minto allow for sufficient time for fibrin digestion by the plasmin.Finally, the remaining plasmin solution was perfused through the channelat the same shear rate. The digested fibrin samples were collected andsnap frozen at −70° C. until assayed for D-dimer.

Scanning Electron Microscopy

After plasma perfusion and the heparin wash buffer wash, the glass slidewith the fibrin deposit was immersed in a glass slide holder containing2.5% glutaraldehyde solution for 5 minutes, then immersed in anotherglass slide holder containing de-ionized water for an additional 5minutes. The slide was then rinsed in graded ethanol solutions (50%,70%, 80%, 100% and 100%) for 5 min, and dehydrated once in 50%, andtwice in 100% hexamethylsilazane for 5 min. Next, a 10-20 nm layer ofgold was sputtered on the dehydrated fibrin deposits. Images were takenwith JOEL 7000 field emission SEM (Hitachi, Tokyo, Japan) at anaccelerating voltage of 1.5kV and a working distance of 6 mm. Thediameters of 20-30 fibrin fibers were measured with Image J software,averaged and reported with standard deviations.

All of the documents cited herein are incorporated herein by reference.

The foregoing description of the present invention has been presentedfor purposes of illustration and description. Furthermore, thedescription is not intended to limit the invention to the form disclosedherein. Consequently, variations and modifications commensurate with theabove teachings, and the skill or knowledge of the relevant art, arewithin the scope of the present invention. The embodiments describedhereinabove are further intended to explain the best mode known forpracticing the invention and to enable others skilled in the art toutilize the invention in such, or other, embodiments and with variousmodifications required by the particular applications or uses of thepresent invention. It is intended that the appended claims be construedto include alternative embodiments to the extent permitted by the priorart.

What is claimed:
 1. A method for evaluating a blood product of anindividual, comprising: a. perfusing the individual's blood product overa microfluidic device under flow conditions to contact the blood productwith a functional molecule of a plurality of coated lipid particles,wherein the microfluidic device, comprises at least one microfluidicchannel; and at least one substrate surface provided in the at least onemicrofluidic channel, wherein the at least one substrate surfacecomprises a plurality of lipid coated particles immobilized on thesubstrate surface, wherein the plurality of lipid coated particlescomprises at least one functional molecule, wherein the at least onefunctional molecule induces coagulation; and b. detecting one or morecoagulation products associated with the at least one functionalmolecule of the plurality of the lipid coated particles.
 2. The methodof claim 1, wherein the blood product is selected from the groupconsisting of whole blood, plasma, platelet rich plasma, and plateletpoor plasma.
 3. The method of claim 1, wherein the flow conditionssimulate hemodynamic conditions of the individual.
 4. The method ofclaim 1, wherein the functional molecule is one or more transmembraneproteins.
 5. The method of claim 1, wherein the transmembrane protein isselected from the group consisting of tissue factor, thromobomodulin,endothelial cell protein C receptor, glycoprotein Ilb/IIIa, glycoproteinVI, glycoprotein 1b/IX/V, P-selectin, glycoprotein IV, CD9, plateletendothelial cell adhesion molecule (PECAM-1), Ras-related protein 1b(rap1b), c-type lectin-like receptor 2 (CLEC-2), intracellular adhesionmolecule 1 (ICAM-1), intracellular adhesion molecule 2 (ICAM-2) andcombinations thereof.
 6. The method of claim 1, wherein the functionalmolecule initiates coagulation.
 7. The method of claim 1, wherein thefunctional molecule inhibits coagulation.
 8. The method of claim 1,wherein the step of detecting comprises quantifying the one or morecoagulation products.
 9. The method of claim 1, wherein the one or morecoagulation products consist of proteins selected from the groupconsisting of thrombin, fibrin, thrombin-antithrombin complex,fibrinopeptide A, fibrinopeptide B, D-dimer, prothrombin fragment 1+2,activated factor X, activated factor V, activated factor VIIIa,activated factor IXa, activated factor XIa, activated factor XIIa,activated protein C, activated protein S, and mixtures thereof.
 10. Themethod of claim 1, wherein the one or more coagulation products aredetected by a method selected from the group consisting of brightfieldmicroscopy, darkfield microscopy, fluorescence microscopy, multi-photonexcitation, second harmonic generation, third harmonic generation,atomic force microscopy, scanning electron microscopy, and absorbance.11. The method of claim 1, wherein the plurality of the lipid coatedparticles comprises a plurality of particles having a hydrophilicsurface.
 12. The method of claim 1, wherein the plurality of lipidcoated particles comprises one or more phospholipid structures selectedfrom the group consisting of phosphatidylserine, phosphatidylcholine,phosphatidic acid, phosphatidylethanolamine, phophoinositides,phosphosphingolipids, and combinations thereof.
 13. The method of claim1, wherein the plurality of lipid coating particles are immobilized onthe substrate surface by a method selected from covalent bonding,electrostatic interactions or hydrogen bonding.
 14. The method of claim1, wherein the at least one microfluidic channel is capable of receivingfluid at a first end of the at least one microfluidic channel andallowing the fluid to flow through the at least one microfluidic channelto a second end of the at least one microfluidic channel.
 15. The methodof claim 1, wherein the at least one microfluidic channel is split intomultiple channels.
 16. The method of claim 1, wherein the at least onefunctional molecule is a tissue factor or a thrombomodulin.
 17. Themethod of claim 1, wherein a flow rate of the individual's blood productis between about 50 to 2600 sec⁻¹.
 18. The method of claim 1, wherein aflow rate of the individual's blood product is between 0 and about500,000 sec⁻¹.
 19. The method of claim 1, further comprising an agentwherein the agent is an anticoagulant agent or coagulating agent. 20.The method of claim 1, wherein the individual is a mammal.